Synthesis, Physicochemical Characterization, Biological Assessment, and Molecular Docking Study of Some Metal Complexes of Alloxan and Ninhydrin as Alterdentate Ligands

A series of transition metal complexes of alloxan monohydrate (H2L1) and ninhydrin (H2L2) have been prepared where metal ions are Fe(III), Co(II), Ni(II), Cu(II), Zr(IV), and Mo(VI). Different microanalytical techniques, spectroscopic methods, and magnetic studies were applied to assign the mode of bonding and elucidate the structure of complexes. All solid complexes are of 1:1 (M:L) stoichiometry and octahedral geometry except nickel (II) complexes exist in a tetrahedral geometry. FTIR spectral interpretation reveals that HL1 coordinates to the central metal ion in a bidentate ON pattern, whereas HL2 behaves as an alterdentate ligand through hydroxyl oxygen and carbonyl oxygen either C(1) = O or C(3) = O. The thermal behavior of some complexes was followed up to 700 °C by different techniques (TGA, DTA, and DSC) where decomposition stages progress in complicated mechanisms and are ended by the formation of metal oxide residue. Besides, biological screening involving antioxidant, antibacterial, and antifungal for ligands and some of their complexes was done. Moreover, four examined metal complexes displayed anticancer activity against hepatocellular carcinoma cells (HepG-2) but to different degrees. According to the IC50 values, Cu-ninhydrin complex, [Cu(HL2)(H2O)4].Cl has a better potency impact in comparison with cisplatin which was used as a reference control. This is in harmony with the molecular docking simulation outcomes that predicted a good binding propensity of the Cu-ninhydrin complex with hepatocellular carcinoma protein (2jrs). Therefore, the Cu-ninhydrin complex should be deemed as a potential chemotherapeutic agent for hepatocellular cancer. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s10904-023-02661-5.


Introduction
Alloxan and ninhydrin are privileged compounds because of their inclusive range of applications in biological and organic synthesis fields [1][2][3][4][5]. Alloxan is considered an example of a pyrimidine derivative while ninhydrin possesses an indanone core moiety that is found in many natural products. Detailed investigations of these compounds are essentially important to correlate the relationship between their structures and their biological efficacy [6,7]. As well, pyrimidine derivatives are known for many diverse applications as antioxidants, anti-inflammatory, and antiviral drugs [8,9]. Especially with the current coronavirus threat, many research efforts are converging to find more effective antiviral agents by making different changes in the pyrimidine building block [9]. In our group, a series of papers have been reported to throw light on the chemistry of the biologically active pyrimidine derivatives and their metal complexes [10][11][12]. Also, many efforts have been done to apply ninhydrin in fluorescence techniques by developing new fluorogenic systems such as the human serum albumin assay [13]. Some ninhydrin-based fluorophores were also studied to behave as selective ligands for certain metal ions [14].
For decades, there have been a growing number of research studies on the complexation of alloxan since it possesses more than one equivalent chelation site as a good example of alterdentate ligands [15][16][17]. Possible metal ion exchange between these sites occurs via intra-or intermolecular pathways [18]. Nevertheless, the involvement of ninhydrin in the isolation of mononuclear metal complexes is rare in the literature [19,20]. Besides, there is increasing attention to designing and developing novel non-platinum metal complexes to overcome the unsolved clinical problems combined with platinum-based chemical drugs [21,22]. In continuation, the main objective of this research aims to prepare a series of metal complexes derived from alloxan or ninhydrin for diverse applications in the hope of innovating low-cost anti-pathogenic or anticancer candidates and GAUSSIAN 09 software was used for the calculations of molecular orbital parameters based on the DFT(B3LYP/6-31G) level of theory. The optimized structures were visualized in GAUSSIAN-VIEW. Docking investigation was attained by MOE 2015.10 software. The initial steps to prepare the tested compound for the docking process included hydrogen atoms addition, removal of water molecules, atomic charges clarifying, and then energy minimization by MMFF94x force field [26].

Antimicrobial Activity
Screening tests regarding the in vitro inhibition zone measurement in mm were carried out by the well diffusion method [27]. Briefly, the inoculum suspension was prepared from colonies grown overnight on an agar plate in 10 ml of Mueller-Hinton agar medium (Merck, Germany). A sterile swab was immersed in the bacterial suspension and used to inoculate agar plates. Each tested compound was dissolved in dimethyl sulfoxide (DMSO, 5 mg/ml). 100 µl was tested and each inhibition zone was measured around each well after 24 h at 37 °C. Controls using DMSO were done, and tests were duplicated by using ketoconazole and gentamycin as references for antifungal and antibacterial activities, respectively.

Antioxidant Activity
The stable radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) was used as a reagent for spectrophotometric assay. 200 µl serial concentrations of each sample (150, 300, 450, 600, 750, 900, 1000 mg) were mixed with 1 ml of DPPH (0.0025 g/ml in methanol), each separately. The mixture was shaken vigorously and then kept in dark for half an hour, the decrease in absorbance of each mixture was measured spectrophotometrically at 517 nm. Blank was prepared in DPPH without antioxidants, while vitamin E was used as a synthetic reference [28]. The effective concentration of a sample that needed to scavenge DPPH radical by 50% (EC 50 value) was obtained by linear regression analysis of doseresponse curve plotting between % scavenging and concentrations. Antioxidant tests were performed at the biochemistry laboratory, Faculty of Science, Alexandria University, Alexandria, Egypt.

Anticancer Activity
Four of the synthesized metal complexes were evaluated as a preliminary study for their human tumor cell growth inhibitory activity against hepatocellular carcinoma cells (HepG-2). Cytotoxicity evaluation using crystal violet viability assay was accomplished as described [29]. Cisplatin was utilized as positive control under the same measurement conditions. Antimicrobial and cytotoxicity evaluations were carried out by Regional Center for Mycology & Biotechnology (RCMP) at Al-Azhar University, Cairo, Egypt.

General Procedure for the Synthesis of Metal Complexes
All complexes were prepared in a similar procedure. The required weight of the transition metal chloride or sulfate salt (2 mmole) was dissolved in 10 ml distilled water and mixed with the required weight of ligand (2 mmole) dissolved in 25 ml methanol. Then, drops of ammonia solution (1:1) were added to make the medium slightly basic (pH 8.0) except in the case of iron complexes. The reaction mixture was heated to 60 °C for 1 h with continuous stirring, then cooled where a precipitate was formed, filtered then dried in an oven at 90 °C.

Characterization of the Synthesized Metal Complexes
Characterization of the investigated compounds was elucidated by different microanalytical, spectroscopic tools, magnetic susceptibility, and thermal studies.

FT-IR Spectra of Ligands and Their Complexes
Detailed interpretations of infrared spectral data with some important characteristic assignments are elaborated, Figs. 1, 1S, 2S and 3S and Tables 1S, 2S in the supplementary electronic information. The infrared spectra of the prepared complexes were compared with that of the free The IR spectra of alloxan and its metal complexes exhibit strong broad bands in the range of 3448 − 3041 cm -1 , Figs. 1 and 1S, corresponding to overlapping between ν(OH) and ν(NH) vibrations [15]. The broad feature of the bands in this region could be taken as evidence of the coordination with H 2 O molecules and the presence of intramolecular hydrogen bonds [17]. This is convenient with the analytical data and TGA results where all complexes possess some inner sphere water molecules. Also, intense carbonyl bands appeared in the alloxan IR spectrum  (4) with similar H-displacement as alloxan is an alterdentate ligand as mentioned before [18]. Besides, The fundamental vibration bands of ninhydrin and its metal complexes are shown in Table 2S and Figs. 2S and 3S. The aromatic ν(C-H) stretching appears at wavenumber 3089 cm -1 while δ(C-H) bending modes are observed at 1255, 1185 and 1065 cm -1 (in-plane), and 740 cm -1 (out of plane) [31]. The broad band at 3300 cm -1 in the spectra of free ninhydrin due to ν(OH) is shifted in the spectra of metal-ninhydrin complexes suggesting coordination through the oxygen atom of one of the hydroxyl groups in the prepared complexes. Also, the infrared spectrum of ninhydrin exhibits the ν(C = O) at 1749 cm -1 which suffers an obvious lowering in intensity in the spectra of its metal complexes. This negative shift indicates the involvement of one of the two carbonyl groups of ninhydrin in coordination with the metal ion.
Likewise, the appearance of the new band at 520-472 cm -1 in the spectra of all metal-ninhydrin complexes, which is absent in the spectra of free ninhydrin ligand, can be assigned to ν(M-O) [30]. This reveals that ninhydrin can react with metal ions as a bidentate ligand involving hydroxyl oxygen and carbonyl oxygen either C(1) = O or C(3) = O since ninhydrin is an alterdentate ligand as alloxan [18].
Notably, Mo-alloxan and Mo-ninhydrin complexes, Figs. 1S and 3S, display a strong band at 937 and 935 cm -1 , respectively, that is designated to ν(Mo = O) confirming the presence of such bond in their structures,

Electronic Spectra, Magnetism, and Conductivity Measurements
The UV-Vis spectra of the ligands (H 2 L 1 and H 2 L 2 ) and their metal complexes are collected in Table 2; Figs. 4S and 5S. The electronic absorption spectra of the iron complexes of the entitled ligands showed two bands at 270-275 and 345-350 nm due to π → π* and CT (L→M), respectively. The room temperature µ eff values of 2.21-2.22 B.M typified the existence of octahedral configuration in a low spin state [33]. The structure of these iron (III) complexes is constructed depending on the bidentate nature of alloxan or ninhydrin ligands with the existence of four water molecules in the inner sphere, and one SO 4 2− group in the outer sphere, Figs. 2 and 6S.
The electronic spectra of cobalt complexes showed three bands. The first two bands in the ranges 260-280 and 300-320 nm could be assigned to π → π* and LMCT in that order. Nevertheless, the third weak intense band at 380-386 nm is due to the 4 T 1g (F) → 4 T 1g (P) transition typifying octahedral geometry. The room temperature µ eff value of 5.02-5.03 B.M implied a high spin environment for the two complexes with weak coordination bonds [33]. However, the electronic absorption spectra of the Ni-alloxan complex showed two bands at 290 and 480 nm due to π → π* and 3 T 1 → 3 T 1 (P) transitions. The room temperature µ eff values of 4.11-4.12 B.M, Table 2, typified the presence of the two nickel complexes derived from alloxan and ninhydrin in perfect tetrahedral geometry [34].
The electronic absorption spectra of the two copper complexes showed three bands in the range of 260 to 385 nm. The broadening of the third CT band expands to 460 nm inside the visible region. The broad feature of this band makes it difficult to distinguish any weak d-d transitions in this region [22]. The observed values of µ eff for copper (II) complexes are 2.30-2.31 B.M which is higher than the spin-only value corresponding to one unpaired electron 1.73 B.M. This could be attributed to the orbital contribution to the spin of octahedral complexes [33]. The Oh structure is established by the bidentate mode of the ligands and four water molecules in the inner sphere with the presence of one chloride ion in the outer sphere, Figs. 2 and 6S.
As for all d 0 configurations, no characteristic d-d transitions are expected for Zr 4+ and Mo 6+ ions in visible light > 380 nm. Any found bands at lower wavelengths (in the UV region) could be assigned to CT transitions of the type L → M, or π→π * and n → π * transitions of the organic part of the complexes [35]. The diamagnetic feature of zirconium (IV) and molybdenum (VI) complexes proposed that these ions exhibit octahedral coordination as similarly reported complexes [32,35].
The molar conductance (Λ m ) of the isolated complexes was determined. The measurement was done in ethanol of 10 -3 M solution at 25 °C. The Λ m values for Mo and Zr complexes are comparatively low (9.7-19.8 Ω -1 cm 2 mol -1 in the case of alloxan complexes), signifying the non-electrolytic manner of these complexes, Table 2 [36]. However, the molar conductance is of relatively higher values for Fe, Co, Ni, and Cu complexes (49.5-207 Ω -1 cm 2 mol -1 in the case of ninhydrin complexes), supporting the electrolytic property of these complexes and the existence of SO 4 2− and Cl − ions in the outer sphere, Figs. 2 and 6S [37]. It is worth mentioning that different complexes of alloxan (H 2 L 1 ) have been isolated in previous studies [16,17]. For instance, alloxan was bound to Fe(III) as a dibasic bidentate ligand [17], [Fe(L 1 )(H 2 O) 3 4 ].SO 4 , as in our case. This could be attributed to the variation in the pH of the medium during the synthesis process and the type of counter ions (Cl − or SO 4 2− ) of the used metal salt [22,38]. using Eyring equations and Coats-Redfern method [39], Table 3S, associated with each degradation step,   Table 3. The small discrepancy between the calculated and found weight losses in the second and third stages is taken as an indication of overlapping of the decomposition steps in these stages. It is worth stating that the formation of these residues at such a high temperature range (700 °C) could be taken as an indication of the higher thermal stability of the studied complexes.
The thermal decomposition of the ninhydrin ligand takes place in two stages of mass loss, Fig. 9S. The first step progresses up to 189 °C which is due to the loss of 2 H 2 O molecules and corresponds to the first endothermic DTA peak at 146.6 °C. Besides, the second endothermic DTA peak at 261.7 °C allocates the ninhydrin melting point [40]. After melting, decomposition continues at higher temperatures until the removal of the rest of the compound as gaseous molecules with almost no residue.
Regarding DTA curves, the first peak of the DTA curve of The positive sign of the enthalpy (∆H*) and the free energy (∆G*) of activation in all studied cases, Table 3S, reveals that all decomposition steps are of endothermic and nonspontaneous nature, respectively, where the final residue is of higher energy than that of the started complex. Further, the values of (∆G*) increase considerably for the successive decomposition stages for each compound. This causes an increase in the removal rate of the precedent species than that of the subsequent one [41]. Furthermore, the entropy of activation (∆S*) is of negative value for all decomposition processes implying that the activated complexes are of a more ordered structure compared to the undecomposed complexes [42]. Also, the values of ∆S* are nearly equal, Table 3S, so the reaction was entropy-independent.
DSC is also used to study thermal transitions such as glass transition (T g ), melting (T m ), and crystallization (T g ) temperatures [43], Table 4 S and Figs. 5 and 11 S. The glass transition temperature exhibits a dehydration process of the inner sphere water molecules followed by thermal agitation decomposition. This is in harmony with the TGA findings for these complexes. All the prepared complexes melt above ∼ 250 °C. DSC plot is used to determine the melting temperature through an endothermic transition, where the compounds should absorb heat until all the crystals are melted. T m values of ligands and their complexes are varied between 255 and 450 °C, Table 4 S.

Structural Optimization Using DFT
Density functional theory (DFT) was employed to calculate the molecular parameters of the best optimized conformations of the studied ligands (H 2 L 1 & H 2 L 2 ) and some of their metal complexes. DFT is considered a reliable method to identify the nucleophilic and electrophilic sites as well as the molecular polarity that affects their reactivity with metal cations or biological receptors. The structure of the ligands and their Cu(II) and Fe(III) complexes as illustrative examples were optimized to the lowest energy conformers with DFT-B3LYP/ 6-31G basis set using Gaussian 09 program [44,45]. The theoretical data of the investigated compounds are collected in Table 4; Figs. 6, 7, 8, 9 and 12S-17S.
Generally, a small HOMO-LUMO energy gap (ΔE) is taken as a good indication of softness, chemical reactivity, and the complexation ability of ligands [46], Fig. 7. Ninhydrin and alloxan exhibit smaller ΔE values (4.315-5.004 eV) pointing to their capability to offer electrons to vacant d-orbitals of any neighboring central metal ion [47]. Also, the higher negative total energy (E T ) of Cu(II) and Fe(III) complexes compared to their free ligands, Table 4, suggests the greater stability of prepared complexes [22].
In general, there is a direct correlation between biological potency and chemical reactivity indices such as dipole moment (D), hardness (η), and softness (S). Accordingly, the Cu-alloxan complex shows the lowest η (0.706 eV) and highest S (0.708 eV − 1 ). So, it is expected to have an adequate amount of softness and hence superior biological activity as proven practically in this research. Likewise, the estimated values of η (1.005 eV) and S (0.498 eV − 1 ) of Cu-ninhydrin predict its biological potency as confirmed later in the anticancer study section. The electrophilicity index (ω) describes the reactivity of various specific sites on the surface of the molecule, as it measures the stabilization energy when the system acquires an additional negative charge from the environment. It also quantifies the biological activity of drug-receptor interactions.
One of the validated approaches to evaluating the structure-activity relationship (SAR) is to study the electron density distribution on the surface of bioactive molecules. This allows for estimating the lipophilic capacity and hence feasible ability to penetrate the phospholipid bilayer of the cell membrane. It is well known that the cell membrane of almost all microorganisms is a phospholipid bilayer. In general, compounds of low dipole moment (D) are only capable of diffusing through this type of membrane as the lipophilicity is inversely proportional to the polarity degree [48]. Cuninhydrin has the lowest dipole moment value (2.52 Debye) and so it is expected to have good biological efficacy.
The molecular electrostatic potential (MEP) of alloxan (H 2 L 1 ) and ninhydrin (H 2 L 2 ) ligands were constructed theoretically to give an idea about the electronic charge distribution on their surfaces specifying the nucleophilic and electrophilic sites for possible chemical interactions, Fig. 8.

Molecular Docking Study
In the recent two decades, computational simulations enhanced noticeably making it feasible to utilize computational approaches in drug design. The Molecular Operating Environmental module MOE2015 [49] software package is used to predict the biological features of candidate drugs and to anticipate the experimental results. In the present study, the protein structure of hepatocellular carcinoma (2jrs) [50] was used as the receptors docked with alloxan, ninhydrin, and their Cu(II) & Fe(III) metal complexes (inhibitors). Before the docking process, the preparation of the preprotein structure was achieved by removing water molecules and adding polar hydrogens with the MMFF94x force field. The calculated data for the docking process between all tested compounds (ligands) and the selected protein 2jrs (receptor) are listed in Table 5; Figs. 9 and 16S, 17S.  Docking results include ligand-receptor sites, interaction type, interaction distances (Å), internal energy (E), and scoring energy (S) in kcal/mole. The negative value for energies implies the spontaneous binding of the tested inhibitor to the target protein. The data propose the best interaction stability for docked compounds. The effective ligand-receptor interaction distances were ≤ 3.5 Å in most cases, Table 5, which indicates the presence of typical real bonds and hence high binding affinity [26]. For example, the nearest interaction is observed via H-donors with Aspartate ASP 77 (2.62 Å) and Proline PRO 26 (2.68 Å) amino acids in the case of Fe-alloxan and Cu-ninhydrin complexes, respectively, Figs. 9 and 17S. Further, the scoring energy function (S) is taken as an indication of high ligand-protein binding affinity based on several factors such as hydrogen bonds, deformation impact, hydrophobicity, entropy, and van der Waals interaction [22]. Furthermore, five and six binding sites were observed with different amino acids in the case of Cu-ninhydrin and Fealloxan complexes with relatively higher negative scoring energy (− 3.9133 and − 3.9075 kcal/mol, respectively) demonstrating their high inhibition efficacy as candidates against HepG-2 cells.

Antimicrobial Examination
The antimicrobial screening data of the ligands and some of their synthesized complexes were collected in Table 6; Fig. 18S. The tested samples showed negligible to good activities with mean inhibition zone values less than the used standards (ketoconazole for antifungal, and Gentamycin for antibacterial activity). The in vitro antimicrobial evaluation of the studied compounds exhibited varying inhibitory effects. Based on these data, alloxan showed no antibacterial effect against Staphylococcus aureus and Bacillus subtilis, but its iron (III) and copper (II) complexes showed a good inhibition effect against these Gram-positive bacteria. However, the Fe-alloxan complex showed the best antibacterial effect against Escherichia coli and Proteus vulgaris compared to alloxan or Cu-alloxan. Likewise, ninhydrin showed no antifungal activity against Aspergillus fumigatus and Candida albicans, but after complexation, its copper (II) complex showed good antifungal activity against both fungi strains. Also, the Cu-ninhydrin complex showed more antifungal activity (16 mm) against Candida albicans than Fe-ninhydrin. Similarly, Cu-ninhydrin showed more inhibition zone diameter (20 mm) against Staphylococcus aureus than the iron (III) complex. As both Cu(II) and Fe(III) complexes have the same M:L stoichiometry (1:1) and geometry (Oh), it appears that the metal ion type and/or the ionic radius may play an important role in enhancing or quenching the biological activities of these complexes invoking Overtone's chelation theory [21,51]. Seemingly, the activity in most cases is increased upon chelation where the positive charge of the metal is partially

Antioxidant Property
A lot of research effort has been focused to design new radical-scavenging compounds as effective antioxidants to control the phenomenon of oxidative stress that is produced by the imbalance between the accumulation of reactive oxygen species (ROS) in body tissues and the ability to remove potentially harmful free radicals. This accumulation of free radicals in cells can cause large chain chemical reactions and pathogenic mechanisms leading to many serious diseases such as rheumatoid arthritis, atherosclerosis, and cancer propagation [53].
The examined compounds have shown scavenging activity with different values regarding their distinct structures, compared to vitamin E as an antioxidant reference using the DPPH assay method [28], Fig. 10. Interestingly, the estimated EC 50 value of Cu-ninhydrin complex was found to be 320 (µg/ml), which is lower than the EC 50 of vitamin E (400 µg/ml), and hence it is expected to possess a promising antioxidant effect.

Anticancer Activity Assessment
The in vitro cytotoxicity activities of some synthesized metal complexes have been evaluated against a human cancer cell line (HepG-2) compared to cisplatin as a standard anticancer drug, using viability assays as described in the experimental part. The results are collected in Table 7; Figs. 11 and 12. Based on cytotoxicity results, the four examined complexes have different anticancer activity against hepatocellular carcinoma cells (HepG-2) according to IC 50 values. Copper complexes of alloxan and ninhydrin are more effective against hepatocellular carcinoma cells than iron complexes of the same ligands. While the copper complex of ninhydrin (IC 50 = 3.01 ± 0.3 µg/ml) is more effective against HepG-2 cells than the copper complex of alloxan (IC 50 = 26.5 ± 1.8 µg/ ml). Also, the Cu-ninhydrin complex has an even better potency impact, Fig. 12, in comparison with cisplatin (IC 50 = 3.68 ± 0.19 µg/ml) which was used as a reference  The inhibitory dose curves of Fe-alloxan and Cu-alloxan compared to cis-platin against HepG-2 cell line control for anticancer activity. Notably, the very low IC 50 value for Cu-ninhydrin complex (3.01 µg/ml = 8.64 µM) is virtually under the authorization of the US NCI program that if the IC 50 value is less than 10 µM after the 72-h incubation period, the tested compound is assumed to have in vitro promising antiproliferative action against the examined cell lines [54]. The preceding finding is in harmony to great extent with the molecular docking simulation outcomes that predicted a good propensity of Cu-ninhydrin complex to bind with the liver cancer protein (2jrs) as discussed earlier.

Conclusions
In this research, a series of transition metal complexes of alloxan and ninhydrin were prepared and characterized by different analytical, spectroscopic, and magnetic studies.
All solid complexes are of 1:1 (M:L) stoichiometry and octahedral geometry except nickel (II) complexes exist in a 4-coordinate structure. Density functional theory (DFT) is used to construct the optimized structures of the ligands and their molecular electrostatic potential surfaces. Further, the two ligands and four of the designed complexes were subjected to molecular docking simulation as a preliminary SAR study. Biological screening of some compounds was tested as antioxidant and antimicrobial (against four grampositive & gram-negative bacteria, and two fungi strains). Compounds with noticeable activity may be considered as a starting point for the development of some new therapeutic drugs. Some examined metal complexes display anticancer activity against hepatocellular carcinoma cells (HepG-2) but to different degrees. According to the IC 50 values, the Cu-ninhydrin complex has a better potency impact compared with cisplatin. This is in good correlation with the molecular docking simulation outcomes that predicted a strong binding of Cu-ninhydrin complex to the liver cancer protein (2jrs). So, the Cu-ninhydrin complex could be considered a potential candidate as a chemotherapeutic agent for hepatocellular cancer after further clinical studies such as ADMETscore, intracellular ROS generation, clinical trials, and drug approval processes. Besides, the copper (II) complex derived from ninhydrin is economically expected to be of low cost to other platinum-based chemical drugs.  otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.